Production and use of high pressure for cryopreservation and cryofixation

ABSTRACT

Methods and devices are described for the concurrent delivery of elevated pressures and low temperatures to a sample, typically but not exclusively a biological sample. A medium that expands on cooling and/or freezing is employed with a sample immersed therein, typically but not exclusively encased in a sample container. Cooling the medium lowers the temperature and applies pressure to the sample such that reduced damage to a typical biosample occurs. Relatively long-lived metastable phases are also produced, including both metastable liquids and solids, without the need for very rapid cooling steps as required in conventional achievement of such metastable phases. Preliminary test data are also presented.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a utility patent application filed pursuant to 35 U.S.C. §111 (a), and claims priority pursuant to 35 U.S.C. §119 from provisional patent application 61/463,727 filed Feb. 22, 2011. The entire contents of the aforesaid provisional patent application is incorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the general field of the production of use of high pressures applied isostatically to a sample or workpiece, more particularly to high pressures in combination with low temperatures for the cryopreservation or cryofixation of biological samples.

2. Background and Related Art

An important challenge affecting many aspects of medical, biological, chemical, agricultural and food sciences is the long-term storage and preservation of various substances whose properties tend to degrade over time. Even when storage procedures exist and are in common use (e.g., refrigerated storage of blood for transfusion), improved storage methods are continually being sought. Examples abound, spanning the range of sample sizes from molecules to organs and, indeed, entire organisms.

Many individual molecules degrade in storage, particularly troubling for pharmaceuticals, losing efficacy and possibly degrading into substances harmful for the patient. Increasing shelf life for storage and transport of these chemicals is an important practical goal.

Biological substances (“biosubstances” or “biosamples”) deriving from living or formerly living organisms must frequently be stored in a non-degrading manner such that reanimation and/or reuse is feasible. For economy of language, we indicate such storage of biological substances in a condition permitting reanimation and/or reuse as “viable preservation,” and, when low temperatures are employed, “viable cryopreservation.” Blood and other biological fluids, tissues, organs, embryos, groups of organs are among those biosubstances for which viable preservation is, or potentially could be, a significant medical procedure bringing valuable advances in treatment options.

Viable preservation of biosubstances also has an influential role to play in agriculture, aquaculture and animal breeding. Perhaps the most conspicuous present application relates to the viable preservation of semen for cattle breeding. However, improved preservation techniques for embryos is also likely to have significant effects in these fields.

Viable preservation should not be overlooked for its potential impact on food sciences. In this case, “reanimation” relates to the recovery of the preserved biosubstance in a form suitable for its intended use, that is, consumption as a food product. Many food products (such as fresh strawberries) do not generally appeal to consumers when thawed following conventional food preservation by freezing. A viable preservation technique suitable for use in the food industry may also bring notable practical benefits and new products.

Freezing biological samples is also an important technique in the preparation of samples for microscopy, particularly electron microscopy. Freezing a biological sample (“cryofixation”), perhaps in combination with staining or fixation agents, can produce a solid from which thin sections can be sliced suitable for use in electron microscopy. In addition, freezing followed by mechanical cracking of the sample (“freeze fracture”) can expose features of the three-dimensional structure of the biosample that can be imaged in an electron microscope. Evaporation of a suitable imaging agent at an angle onto the fractured surface of the sample can enhance the view of the three-dimensional structure. However, it is important in cryofixation that the freezing process not substantially disrupt the structures sought to be studied microscopically, a serious problem with many conventional freezing techniques.

Thus, a need exists in the art for improved methods for the preservation of biosamples in a condition for reuse following preservation, including improved equipment for performing such preservation.

BRIEF SUMMARY OF THE INVENTION

One important objective of the methods described herein is to describe a method of cryogenically storing a sample so as to reduce sample degradation, accomplished in some embodiments by the concurrent isostatic application of elevated pressure while cooling the sample. This is typically accomplished by immersing the sample in a working fluid (advantageously encased in a sample container in most instances) wherein the working fluid expands upon cooling; while confining the working fluid in a pressure-confining container such that expansion of the working fluid will result in elevated pressure being applied to said sample; and, cooling the pressure-confining container thereby causing elevated pressure to be applied to the working fluid and the sample. In many practical cases, water is advantageously used as both the pressure-transmitting fluid and the working fluid although other fluids might be used as well as various solvents containing various solutes dissolved therein, as would be apparent to those having ordinary skills in the art and/or readily discoverable with routine experimentation.

We also describe how the methods described can lead to the production of relatively long-lived metastable supercooled liquid but without the necessity of an ultra-rapid cooling step.

We also describe how the methods described can lead to the production of a state of matter substantially similar to high density amorphous water without the necessity of an ultra-rapid cooling step. Further cooling can be used to achieve a high density amorphous glassy state without the sudden, significant and often damaging density change between liquid and high density amorphous glass that is a frequent problem in other techniques such as “flash cooling.”

Accordingly and advantageously, these and other advantages are achieved in accordance with the present invention as described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. The drawings herein are schematic, not to scale and the relative dimensions of various elements in the drawings are not to scale.

The techniques of the present invention can readily be understood by considering the following detailed description in conjunction with the accompanying drawings, in which:

FIG. 1: A schematic, block diagram depiction of a typical apparatus.

FIG. 2: Block diagram, schematic depictions of a typical apparatus at various stages in the process.

A. Preparation

B. Cooling

C. Biasing for HDA

FIG. 3: A schematic, block diagram depiction of a typical apparatus with multiple sample chambers in a single pressure vessel.

FIGS. 4, 5, 6: Block diagram, schematic depictions of other representative apparatus showing additional separators and rupture discs.

FIG. 7: Pressure-Temperature of water showing target path.

DETAILED DESCRIPTION

As noted above, it is important in cryopreservation and cryofixation that the freezing process not substantially disrupt the structures sought to be utilized subsequently, either reanimated, consumed or studied microscopically. In particular, normal ice formation is known to disrupt subcellular structures, potentially destroying viability and/or destroying the morphology sought to be imaged. Therefore, many cryofixation procedures seek to avoid ice formation, and seek to produce less disruptive glassy phases. Conventional techniques to form such glassy phases require rapid cooling rates to achieve glass-forming temperatures rapidly (typically around −140 deg. C.), intending to cause the sample to pass through the dangerous ice-forming temperature region too quickly for damaging ice to form. The relatively poor thermal conductivity of typical biological samples thus requires thin samples. In addition, the large thermal gradients or thermal discontinuities typically caused by rapid cooling tends to create glassy samples having undesirable cracks in the solid phase. Hence, it would be advantageous to be able to form glassy phases without the requirement of rapid cooling, thus allowing thicker samples to be subject to cryofixation and microscopic studies. The present techniques offer advantageous approaches to cryofixation as well as cryopreservation, as is apparent from the descriptions herein.

It is also desirable in many cases to be able to observe living samples over a period of time, as well as observing samples that degrade with time, but movement and degradation may impede the resolution of existing microscopy techniques. The ability to preserve and reanimate samples would offer benefits over cryofixation in such microscopy applications.

In view of the above substantial practical benefits, viable preservation has been the subject of an overwhelming amount of research for many decades. To be concrete in our descriptions, we focus on the important practical cases of preservation of tissues and organs. Other applications, such as molecular storage or the preservation of biological fluids will be apparent to those having ordinary skills in the art. Particular distinctions will be noted when appropriate.

It is well known that at low temperatures, chemical reactions in biological systems typically slow down and, for virtually all practical purposes, cease entirely when the biosubstance solidifies. Thus, straight-forward freezing followed by thawing provides adequate long-term storage for some substances (e.g., a steak intended for human consumption), but has not succeeded in preserving for reanimation tissues, organs, etc. that must be reanimated so as to recover an adequate level of biological function. A precise meaning for “adequate” depends on the particular case under consideration. For example, a smaller survival rate can be tolerated among a plentiful sample of sperm intended for fertilization, in which the individual sperm are seemingly indistinguishable and readily recollected. Contrast such samples with scarce samples of patient-biocompatible tissues or organs intended for transplantation, trace forensic or paleontological samples, and the like. In general, samples that are available in quantities greatly in excess of the amount needed to achieve the desired biological function are expected to be more tolerant of losses during processing than unique samples or samples available in adequate but not abundant amounts. For example, improved yields in the preservation of bone marrow have the potential for revolutionizing bone-marrow cancer therapy.

It is helpful in designing a research plan to improve the viability of cryopreservation techniques to understand the mechanisms of damage resulting from cryopreservation. For example, changes in the environmental conditions surrounding a biosubstance can include changes in applied temperature, applied pressure, compositions of surrounding solvents and/or ionic solutions, among others. Some of the mechanisms leading to damage include the following: (a) Damage resulting from excessive solute concentration as solidifying water tends to exclude solutes from the solid phase, concentrating them in the remaining liquid; (b) Osmotic toxicity in which changed conditions and/or solidification as in (a) alter the osmotic concentrations of species on different sides of osmotic membranes, often damaging the biosample in the process; (c) Damage to cell walls (membranes) as a result of inter- and intra-cellular water joining ice crystals through cell walls (membranes) before the walls (membranes) have solidified sufficiently, among others.

It is well known that such changes as noted above can affect numerous chemical and physical processes within biosubstances as well as displace chemical and physical equilibria (e.g., membrane transport). However, viability of a particular cryopreservation process would only be affected by such changes that are irreversible, that is, cause damage to the tissue, cellular or subcellular structures that do not repair themselves when the environmental conditions are returned to normal. On this basis, the chief cause of irreversible damage occurring during cryopreservation seems to revolve around the formation of deleterious solid phases. It is conventional to refer to such solid phases as “ice” without regard to the detailed microstructure(s) present. Nevertheless, it is useful to consider damage to biosubstances from formation of solid phases (or avoidance thereof) in terms of the formation of various solid phases of water, crystalline, amorphous, among others. For economy of language we will follow this convention, using “ice” as a generic term for solidified water primarily composed of one or more crystalline phases of water, understanding thereby that it is a simplification in the complex solution environment of a biosubstance.

Three major classes of irreversible damage from ice formation during cryopreservation have been identified. (1) Structural damage to cellular or subcellular features by ice formation. (2) Disruption of osmotic or other equilibria balances by the selective concentration of specific substances in the as-forming solid phase (or in the unsolidified remaining solution). (3) In some tissues, particular damage resulting from the disruption of connective or anchoring structures, typically damage to multicellular structures in contrast to cellular or subcellular structural damage.

Thus, efforts to improve cryopreservation have largely focused attention on the damage expected from the formation of ice or similar solids: suppression thereof, changing temperature or other conditions of ice formation so as to cause ice formation to occur in less damaging regions, mitigating the deleterious effects of ice formation if and when it does occur, among other approaches to damage avoidance and/or mitigation. Three general approaches have been employed, often in combination, with a nearly limitless range of variations in each.

1) Temperature Protocol: Adjusting the cooling rate to suppress ice formation to as low a temperature as possible. This typically requires careful tailoring of the time-temperature protocol for the particular system of interest, making it challenging to implement for tissues, organs or collections of more than a few cells for which heat transfer is generally poor resulting in hard-to-control temperature variations throughout the sample.

2) Additives to Suppress Ice Formation: Additions of such “cryoprotectants” function as a species of anti-freeze, avoiding solidification of the biosubstance until much lower temperatures are reached than would typically result in ice formation. Unfortunately, many cryoprotectants are poisonous to living cells, tissues or organs so can be used in only modest amounts for relatively short periods of time. However, cryoprotectants can also be hazardous or dangerous to non-living biosamples or to chemical compounds. For example, DNA/RNA strands or other biochemicals can be affected in the presence of cryoprotectants, as can the structures and/or chemistry of formerly living biosamples, possibly degrading their usefulness for subsequent purposes (forensic, research, among others). In addition, it is quite difficult to achieve the desired concentration of cryoprotectant everywhere throughout a “bulk” biosubstance (typically larger than a few cells), practically limiting the application of this technique to samples of single cells or small collections of cells.

3) Formation of Less-Damaging Solid Phases: Many non-crystalline phases of water and solutions have been studied including “vitreous”, “amorphous”, among others, with numerous sub-types within these general classes of water or water-like solids, chiefly to achieve less damaging non-expanding solids, but other motivations may be present as well. It is desired to cool the biosubstance under consideration to a solid state (thereby stopping all chemistry and biology) but form a solid phase that results in reduced amounts of irreversible damage. Numerous procedures have been investigated for achieving this end including very rapid cooling rates, vitrification facilitated by added cryoprotectant-like substances, use of elevated pressures, among others. The present invention generally relates to techniques for avoiding and/or reducing the formation of deleterious solid phases as specified in more detail below.

Achieving cryopreservation with only acceptable amounts of damage to the biosubstance is just the first part of a successful viable cryopreservation protocol. The cryopreserved biosubstance must then be reanimated after some period of storage. Reanimation may be a straight-forward reversal of the cryopreservation procedures, or may present quite different challenges to be overcome. We discuss herein the specifics of reanimation protocols in connection with specific cryopreservation protocols when reanimation is not obvious from knowledge of the cryopreservation process.

Some embodiments of the present invention relate to processes, systems, devices and materials for the cryopreservation and reanimation of cells (or other biosubstances) without the addition of cryoprotectant additives (or “preservatives”) such as dimethylsulfoxide (“DMSO”). To be concrete in our descriptions, we describe the specific cases of cryopreservation and reanimation of cells, understanding thereby that tissues, organs and other biosubstances are not inherently excluded and, in specific cases, may yield improved cryopreservation results by the application of techniques substantially similar to those described herein.

The techniques described herein generally employ elevated pressures applied to the cells during the freezing process in order to inhibit, or suppress to lower temperatures, the formation of deleterious ice crystals. We consider the various phases of water and ice as a useful mental model for describing and striving to understand the benefits and drawbacks of the techniques described herein, recognizing that the actual behavior of cells, their contents and their surroundings, may be rather different under the conditions described. Nevertheless, water has traditionally provided important guidance to describing and understanding the mechanisms of cell damage upon freezing, and we also follow that route.

It is believed that one important mechanism by which ice crystals damage cells arises from the expansion of some phases of ice upon freezing from the liquid. As ice forms and expands on freezing, cellular, subcellular and tissue structures are often irreversibly damaged. Thus, an important objective of some embodiments of the present invention is to develop cryopreservation processes that do not lead to expansion of the solid water phases upon freezing.

However, in connection with the procedures and test results described herein, it is useful to consider that the damaging properties of ice crystals might not derive solely from their expansion but also from their rapid expansion. Similar damage might occur from a sudden collapse, as can be seen in the case of HDA, and the potential for stress fractures. One potential advantage of the techniques describe herein is that by developing a method that slowly applies pressure, we simulate a slow transformation from liquid to solid (since supercooled water takes on the characteristics of HDA as it approaches Tg) and thereby replace rapid expansion/contraction with a more gradual process.

“Vitrification” is understood to be the transformation of water into a glassy state, typically occurring at a temperature below about −138 deg. C. Vitrification can lead to the formation of a phase of water known as High Density Amorphous water (“HDA”). HDA is a metastable glassy phase of water that avoids damage due to expansion since HDA water has a higher density (occupies less volume gram for gram) than liquid water at the same. temperature.

However, conventional techniques for achieving HDA water require very rapid cooling from the liquid phase, typically at a cooling rate of about 105 to 106 deg. C. per sec. Due to the relatively poor thermal conductivity of typical biological substances, this rapid cooling rate can be practically achieved only in small samples (single cells or small groups of cells), or in thin slices of tissue sample. Typically, tissue samples are rapidly brought into contact with a cold surface in a technique known variously as “cryo-slamming,” “contact freezing” or “freeze slamming”.

High Pressure Freezing (“HPF”) relates to performing the freezing process under elevated applied pressures. Typically, application of pressures in the range from about 200-220 MPa (MegaPascals) suppresses the crystallization of ice and decreases the cooling rate required to promote the formation of HDA to as low as about 100 deg. C per sec. While HPF has been used in microscopy for cryofixation of samples to be examined, reversing the process for reanimation of the sample has not been achieved.

While HDA has several attractive properties for long-term preservation and storage of biosubstances, several challenges are presented in its production and use. HDA is a metastable substance typically formed under pressures of about 210 MPa. When the pressure is removed from HDA, it will spontaneously convert to a form of low density amorphous water (“LDA”). There is no clear consensus on the lifetime of metastable HDA once pressure is removed, how temperature might affect the lifetime, and whether the particular mixture of components found in any particular biosample sensitively affects the lifetime of LDA. What is clear, however, is that HDA expands by about 20% when it converts to LDA, thereby physically stressing the sample and reintroducing many of the deleterious effects of ice formation. Thus, it would be an important contribution to cryopreservation to prolong the lifetime of HDA.

The present approach to cryopreservation and cryofixation involves the creation under pressure of a supercooled liquid phase at temperatures above the glass transition (Tg) that experimentally results in substantially less damage to the biosample than the formation of a solid phase, ice crystals or glass. We take the “supercooled liquid phase” to be any liquid below ice formation temperature, roughly −10 deg. C. Pressure can be applied to maintain the supercooled liquid phase for extended periods of time, while cooling to lower temperatures, typically in the range from about −80 deg. C. to the glass transition temperature. Holding the temperature at −80 deg. C. has proved to be a convenient experimental approach in much of the work reported herein but is not an essential limitation. These experimental results at about −80 deg C. (conveniently, dry ice temperature, also conveniently achievable and maintainable with commercial refrigeration equipment) indicate that this supercooled liquid state under pressure is metastable and can remain in the metastable state for long periods of time. The precise lifetime of this supercooled liquid phase has not been determined for various conditions of biological interest, but is apparently sufficient for many cryopreservation and cryofixation processes to be performed. We refer to this as “metastable supercooled liquid” understanding thereby that the precise parameters of its metastability have not been determined.

The experimental results herein demonstrate significantly less damage caused by the present cryopreservation methods, as well as the ability to handle larger samples, in comparison with conventional cryopreservation procedures.

The applied pressure is typically in the range of about 200-210 MPa. An apparatus is employed that uses expansion of a working fluid upon freezing to apply pressure to the biosample. This working fluid is conveniently taken to be water but this is not an inherent limitation as other working fluids expanding on freezing will be apparent to those with ordinary skills in the art. Also, the water used as working fluid must be clearly distinguished from the water present within the biosample sought to be preserved.

Thus, long-term storage of the metastable supercooled liquid does not require the maintenance of high pressure on the sample by means of an external pressure-delivery device. Rather, it is sufficient using this apparatus simply to maintain the temperature sufficiently cold so as to generate sufficient pressure on the sample by the working fluid. A dry ice bath or refrigerator at about −80 deg. C. seems to be sufficient and is convenient as well as inexpensive to maintain.

The particular system described herein applies pressure as a function of the percentage of ice that has formed at decreasing temperatures. That is, the pressure increases from ambient to about 210 MPa as more and more ice forms until we reach the pressure at which no more ice can form due to pressure-induced ice suppression.

For many applications, a metastable liquid phase is not sufficient and a genuine solid phase must be produced, advantageously a glassy phase. However, achieving the glass transition temperature (conventionally thought to be about −138 deg. C. for water at ambient pressure, but perhaps as high as −88 C at 210 MPa) starting from a temperature of about −80 deg C. is a much less challenging problem of heat transfer than achieving the glass transition temperature directly from a cooled sample in saline around 0 deg. C. Thus, metastable supercooled liquid is a useful substance for cryopreservation and cryofixation whether or not a solid, glassy phase is the ultimate goal.

In addition, since water is a relatively poor heat conductor, temporarily maintaining the sample at supercooled water temperatures allows isothermic discontinuities within the water to dissipate prior to the glass transition. This enhanced isothermal continuity can reduce the chances of stress fractures within the amorphous glass in cases where these fractures are the result of subtle temperature variations within the glass structure.

FIG. 7 is a depiction of the pressure-temperature (PT) phase diagram of water as a model system to help fix ideas in connection with the present processes. Water phase diagrams are available in numerous standard references including the website noted on FIG. 7. Since pressure and temperature can be independently controlled, each point on the phase diagram corresponds to a real physical state that can, in principle, be accessed in the laboratory. Thus, any protocol varying pressure and temperature corresponds to a path traced out on the PT phase diagram. However, the time to traverse such a path is not depicted in such a PT phase diagram, including different rates of traverse for different portions of the path as well as time lingering at selected points along the path. As noted herein, the speed of a process can have an important effect on the outcome, tending to exacerbate (or hinder) the formation of cracks or other physical or chemical consequences.

Point A is approximately room temperature and atmospheric pressure. Point C is a desirable state of low temperature, depicted in FIG. 7 at high pressure. Prior cryofixation processes typically make the trip from Point A to Point C in a single direct high-speed jump with no attempt to control the PT path. The processes described herein follow path A-B-C on FIG. 7 in a deterministic manner, providing benefits as described elsewhere herein.

Description of Apparatus for Producing Elevated Pressure and its Isostatic Application to a Sample

The production of high pressure and its application to a sample in an isostatic manner is an important technology for many practical applications including metallurgy, powder metallurgy, materials science, chemistry, biology and biophysics, among others. In particular, the application of high pressure to biological samples is an important component in many biological protocols including fixation of samples for microscopy and electron microscopy, storage of biological samples (“biosamples”) for later analysis or use, preservation of biosamples for later re-activation into an active form, among other useful applications of high pressures. Also, many molecules, particularly pharmaceuticals, may suffer degradation and/or loss of properties when stored for extended periods of time. Increased shelf life may be obtained through the application of high pressures, typically in combination with low temperatures.

To be concrete in our descriptions herein, we focus attention on the production of high pressures and its isostatic application to biosamples. This is by way of illustration and not limitation since those with ordinary skills in the art can readily appreciate other uses and other applications for the isostatic pressures described herein as well as the application of high pressure as described herein to other types of samples.

We use the term “isostatic” herein to indicate the application of pressure in a substantially uniform manner from every direction external to the sample. For example, a pressure-transmitting medium, typically a liquid or other fluid, surrounds the sample to which pressure is to be applied. Application of an external pressure to this fluid, even though applied in a non-uniform manner, typically results in pressure being delivered to the sample in a substantially uniform manner. If the sample has channels, openings or voids (collectively referred to herein as “voids” for economy of language) into which the pressure-transmitting medium can penetrate, pressure will also be applied on the inner surfaces of such voids and the voids are typically not removed by the application of pressure. It is desired in many applications that the voids survive pressurization. For example, in cryopreservation of tissues, organs and other (typically larger) biosamples, the voids are often an integral part of the sample (such as heart, kidney, lung, among others) and, therefore, must survive pressurization if the organ is expected to regain functionality following cryopreservation.

On the other hand, if such voids are present but pressure is desired to be applied only on the external surfaces of the sample containing such voids (e.g., in order to remove the voids to produce a unitary solid), the sample is generally encased in a material impervious to the pressure-transmitting medium before isostatic pressure is applied. This is required in the fabrication of metal parts from powdered metal, in combination with high temperatures. However, encapsulation of powders possibly susceptible to degradation in the presence of pressure-induced heating may also profit from encapsulation. In summary, all such procedures and variants well known in the art are included within the scope of “isostatic” as used herein.

Furthermore, our descriptions herein will pay particular attention to the isostatic application of high pressure as a component of a procedure also involving the application of low temperatures to the sample. Such high pressure cryoprocesses have been particularly important in biological and related fields as high pressure has proven to be one way to reduce or avoid damage typically occurring when freezing biosamples. However, this is likewise by way of illustration and not limitation since the techniques of pressure production and application to samples described herein are not inherently limited to cryoprocesses applied to biological samples.

The techniques described herein relate generally to producing elevated pressure on a sample of interest by means of the expansion or solidification of a pressure-producing fluid in a confined volume also containing the sample to which pressure is to be applied. The sample within the pressure-producing fluid may be in direct contact with the pressure-producing fluid in which case the pressure-producing fluid itself may serve as the pressure-transmitting medium for applying isostatic pressure to the sample. Alternatively, the sample may be encased in a separate sample container or sample chamber to which pressure generated by the pressure-producing fluid is applied. Thus, the sample chamber surrounds the sample with additional fluid delivering the pressure from the walls of the sample chamber to the sample, referred to as herein pressure-transmitting fluid. It is anticipated that the use of a sample chamber and pressure-transmitting fluid offer advantages over direct immersion of the sample in the pressure-producing fluid, and will be described in detail herein.

The physical process under consideration herein relate to a fluid having the property that, under changes of thermodynamic conditions applied thereto (typically cooling), the fluid would expand if the fluid were held at constant pressure. For example, water frozen in an open container (i.e., under constant atmospheric pressure) will naturally expand with the formation of normal ice. Various embodiments of the present invention relate to confining an expansible fluid, typically water, such that the fluid is prevented from expanding, or prevented from expanding to the full extent achievable under constant pressure conditions. For economy of expression, we may refer to the fluid's “expansion,” understanding thereby that for the particular conditions, the “expansion” may be partially or completely hindered by the pressure container such that only partial, minimal or no increase in actual volume of the pressure-producing fluid occurs.

In essence, the present invention relates to an expansible pressure-producing fluid held within a confined volume in a pressure-containing chamber (or pressure chamber) such that, when the fluid is subjected to conditions that would cause its expansion if the fluid were held under constant pressure, the pressure chamber hinders free expansion of the pressure-producing fluid causing the pressure to rise within the pressure chamber. For many cases of practical interest, the pressure chamber will be a rigid structure having sufficient strength to contain the pressure-producing fluid without rupture when maximum pressure is obtained under the particular conditions employed. However, this is not an essential limitation in the production of pressure by means of some embodiments of this invention. The pressure chamber may be expandable so long as the pressure exerted by the pressure chamber back on the pressure-producing fluid is sufficient to cause the desired high pressure to be generated within the pressure chamber. For example, expandable sections of the pressure chamber may be allowed to expand under computer-controlled resistance (back pressure), thereby allowing a variable but controlled time sequence in the generation of high pressure within the pressure chamber.

Expansion of the pressure-producing fluid within the pressure chamber is typically achieved by reducing the temperature of a fluid in which such cooling would result in expansion of the fluid (i.e., lower density) if such cooling were carried out under constant pressure. In many cases of practical interest, the cooling of the pressure-producing fluid is carried out under conditions such that this fluid, if confined at constant pressure, would form a solid (perhaps crystalline, amorphous, glass, allotropic, among others) having a lower density than the fluid from which it formed. By confining such an expansive fluid at constant volume (or at a volume less than the solidified fluid would occupy under constant pressure), pressure will increase on the surfaces of the confining pressure chamber as well as on the surfaces of any sample-containing chamber immersed in the pressure-producing fluid or on the sample itself if no sample-containing chamber is used.

We point out that “constant volume” as used herein does not necessarily imply that the fluid is rigidly confined prior to cooling so that no substantial expansion is possible. Any confinement will produce increased pressure so long as the confinement is to a volume less than that which the fluid will occupy when fully solidified at constant pressure. For example, the pressure-confining chamber, container or vessel utilized herein may have elastic walls, allowing for some expansion of the pressure-producing fluid but exerting sufficient back-pressure on the pressure-producing fluid to achieve the desired pressure on the sample. In another example, the pressure-producing fluid may expand against one or more rupture discs, permitting expansion to a larger volume upon rupture of the rupture disc(s), but a volume still sufficiently confining to produce the desired pressure on the sample. A plurality of rupture discs can be arranged so as to rupture sequentially, allowing step-wise control of the pressure.

For economy of language, we refer to all such pressure-producing processes as occurring at “constant volume” or “constrained volume.”

Cooling an expansible fluid in a constant or constrained volume results in a concurrent decrease in the temperature and an increase in the pressure of the pressure-producing fluid, thus tracing a path on the fluid's Pressure-Temperature (“PT”) phase diagram. In general, this path is expected to be reasonably complex as this expansion-generated pressure affects the particular solid phase(s) that may be formed (if any), in turn affecting the effective density of the pressure-producing fluid, in turn affecting the pressure generated, in turn affecting the phase(s) formed. Providing an apparatus that allows effective control of this coupled sequence of process steps to produce useful and interesting results is among the objectives of the present invention.

As noted, the techniques described herein can be used in connection with any fluid that expands on cooling including, but not limited to, mixtures, solutions, combinations, alloys and other substances. It is advantageous in practice and for convenience of handling that the pressure-producing fluid be normally a liquid at room temperature and atmospheric pressure. Water is the most commonly employed fluid, not limited to pure water but includes water as-delivered, not subject to any particular purification process, as well as various aqueous solutions.

The pressure-producing fluids employed herein expand on cooling. Typically, such fluids expand when cooled to their solid state, that is, frozen. As discussed above, the typical processes employed herein tend to alter pressure and temperature in complex ways, not always leading to the formation of a solid phase. However, it is a useful rule-of-thumb that fluids that expand when frozen typically expand when cooled. Thus, in discussing candidate fluids, it is useful to consider the properties of the normal frozen state at atmospheric pressure with the normal liquid state, understanding throughout that this is not a precise specification of the conditions the fluid will encounter in practice.

In addition to water, pure gallium, bismuth, germanium and silicon are known to expand on freezing. For economy of language, we use “fluid” or “water” to denote substantially pure water or other substance as well as solutions and/or mixtures so long as the solution and/or mixture expands on freezing. That is, “water” includes solutions and/or mixtures such that, at a given pressure, the solid phase of the substance has a lower density than the liquid in equilibrium with the solid under those conditions. It is important to note that this expansion on freezing may not occur at all pressures and, indeed, this pressure dependence is the source of some of the utility for high pressure processing of biosamples.

To be concrete in our descriptions herein, we focus our attention on the cooling of a pressure-producing fluid, typically water, to form a solid, that is solidification. This is by way of illustration, not limitation, as the techniques of producing high pressure relate to the expansion of the pressure-producing fluid, not its transformation into a solid. Thus, “solidification” as used herein may include the case in which the pressure-producing fluid does not achieve a conventional solid phase.

Our primary focus herein relates to the production of elevated pressure by fluids that expand upon cooling and/or freezing. However, solidification and/or expansion can be promoted by heating, as for thermosetting plastics and other substances. The apparatus and procedures described herein can be modified in a straight forward manner to make use of heating rather than cooling to produce elevated pressure, as would be clear to persons having ordinary skills in the art.

It is clear that the processes described herein for producing elevated pressures and its application to a sample are readily reversed to release the applied pressure so long as the thermodynamic change causing the elevated pressure is reversible. For example, freezing of a simple fluid to produce elevated pressure is usually reversible by warming, causing the solid to melt. However, if the thermodynamic change leading to expansion/solidification is not reversible, the descriptions herein apply only to the process performed in the direction producing elevated pressure. For example, substances that “set” in response to temperature changes by cross-linking or other chemical processes, would generally solidify in an irreversible manner. However, it is expected that expansion/solidification upon cooling will be the primary practical application of this technology and is our primary focus herein.

Other approaches to the development and application of high pressure by solidification of expansible fluids include the work of Leunissen, Journal of Microscopy, Vol. 235, Pt. I, pp. 25-35 (2009), US Patent Application Publication 2009/0011505 A1 (Jan. 8, 2009); Rubinsky et al. United States Patent Application Publication US 2007/0042337 A1 (Feb. 22, 2007). Prior approaches to developing increased pressure by freezing typically do not employ a separate sample container, immersing the sample directly in the working fluid, which typically results in loss of over 50% of the sample. Such methods also typically rely on rapid freezing, limiting the sample size. Other differences between this work and the techniques and apparatus described herein will be apparent from the following detailed descriptions.

FIG. 1 is a schematic, block diagram depiction of a typical apparatus pursuant to some embodiments of the present invention. The sample to which pressure is to be applied is placed in closed sample chamber 100. This chamber is indicated as a “tube” in some of the figures herein but this does not imply a circular cross section or any other particular shape for the sample chamber. Various shapes may prove convenient for various purposes and all such are included herein when we refer to the sample chamber.

As noted in FIG. 1, water is the pressure-producing fluid envisioned for use in connection with the particular embodiment depicted in FIG. 1. In this example, the sample in sample chamber 100 is immersed in a sample-containing fluid or “liquid” which serves as the pressure-transmitting medium to an actual biosample, not separately depicted in FIG. 1. The various numerical values and descriptions given in FIG. 1 are typical examples appropriate for water, not excluding other values for other pressure-producing fluids or other values usable with water as the pressure-producing fluid.

Chamber 100 may have multiple walls encasing the sample as obvious modifications of the single-walled container depicted in FIG. 1. Various wall geometries and configurations can be employed so long as the wall(s) permit the transmission of heat and (perhaps) pressure from the surroundings to the sample in a manner and at a rate consistent with the process to be carried out. Typical embodiments of the present invention provide pressure delivery to the sample through end caps, not necessarily through the walls of the sample chamber. Other embodiments may allow pressure to be delivered to the sample through the walls of the sample chamber 100 in addition to (or instead of) pressure delivery through the end caps. All such embodiments are included within the scope of the present invention.

Pressure chamber 110 may contain multiple sample chambers as depicted in FIG. 3 so long as pressure and other parameters remain within desired ranges of uniformity, rates of change, and the like.

It is necessary that chamber 100 have the ability to transfer pressure applied to the exterior of 100 to the sample contained inside chamber 100. One method for doing that is depicted in FIG. 1 in the form of sliding gaskets. Other methods for transferring pressure to the sample can also be employed including a deformable chamber 100, one or more collapsible bellows integrally constructed with chamber 100, rupture disc(s), polymeric, plastic or other deformable materials, among others.

As noted in FIG. 1, the materials and structure of sample chamber 100 and pressure container 110 need to be capable of exposure to the desired pressures without rupture or degradation. However, in the case of sample chamber 100, pressure transferred through the plungers (or other means) to the interior of the sample chamber will substantially equalize the pressure on either side of the walls of the sample chamber 100. Thus, no significant pressure difference is expected on the walls of sample chamber, 100, and they need only have the capability to withstand temporary pressure transients for the time and to the extent necessary for the pressure to equalize inside and outside chamber 100. In addition, the materials comprising the sample chamber 100 and the pressure chamber 110 need to be compatible with the fluid containing the sample in 100 as well as the pressure-generating fluid in 110. That is, chemical interactions are to be avoided between the pressure-generating fluid, the sample containing fluid and surfaces of the apparatus in which they come into contact. FIG. 1 includes typical fluids to be used and pressures to be developed when applying the present apparatus to biosamples in which “C”=degree Celsius, “MPa”=MegaPascal in which 1 Pascal (pressure)=1 Newton (force)/(sq. meter), “PTFE”=polytetrafluorethylene, typically the DuPont brand Teflon.

A typical process for the application of the present techniques relate to low temperature processing of biosamples, possibly including vitrification, that is, the transformation of aqueous-based liquids in biosamples into a glassy solid material. Liquid water is known to be capable of transforming into a glassy state at a temperature below about −138 deg. C. This transformation can lead to the formation of High Density Amorphous water (“HDA”), a metastable phase of water. The expansion of water or similar aqueous phases upon solidification is thought to be an important mechanism causing damage to biosamples upon freezing. Since HDA has a higher density than the liquid from which it can be formed, it is believed that HDA will solidify without deleterious expansion.

Conventionally, HDA formation from liquid water (without formation of harmful expanding solid phases of water), very rapid cooling is employed, typically cooling rates of about 105 to 106 deg. C. per sec. These high cooling rates typically limit the application of HDA to very thin samples since heat transfer through biosamples is not generally efficient.

Alternatively, high pressure can be used to produce HDA at much lower cooling rates than 105 to 106 deg. C. per sec. High pressure freezing (HPF) at an applied pressure of about 200-220 MPa suppresses the formation of crystalline ice phases and decreases the cooling rate required to promote the formation of HDA to as low as about 100 deg. C. per sec. HPF has been used in cryofixation for microscopy but, due to sampling handling issues, no system is currently available for reversing this cryofixation leading to reanimation of the sample. Such a process is depicted in FIG. 2 pursuant to some embodiments of the present invention. We note that FIG. 2C mentions “reversing” the sample chamber. This may, but need not, be carried out by means of a mechanical reversal of sample chamber 100, but is thought to be one convenient and advantageous method.

FIGS. 2 b and 2 c depict and describe techniques for non-uniform cooling of the sample, advantageous under conditions as described in FIG. 2. This non-uniform cooling (or warming) is referred to as “biasing” herein.

FIGS. 4, 5 depict additional embodiments in which one or more rupture discs, shutters or similar pressure separators are employed to divide pressure chamber 110 into separate chamber, pressure isolated from each other until the pressure separator is opened.

FIG. 6 depicts and describes another method for biasing ice formation pursuant to some embodiments of the present invention.

While the figures herein depict biasing from only a single end of the sample tube at a time, this is not an inherent limitation. Two-ended biasing can also be carried out, using different temperatures and/or rates of cooling/warming. However, for simplicity of description, and for simplicity of experimental procedure, only single-ended biasing has been performed in the examples reported herein.

An advantage of the apparatus as described herein is that it permits reasonably high pressures to be obtained and applied to reasonably large sample volumes, all at modest cost for the equipment, much less than typical high pressure equipment.

It is apparent from the figures and the other descriptions herein that the present apparatus provides substantial flexibility for testing various processes. For example, the sample chamber 100 can be immersed in the coolant to various depths, for various times and inserted and/or withdrawn according to any desired time profile. The sample chamber need not be immersed in the coolant in any particular orientation, allowing various cooling profiles of the sample to be achieved. The coolant can have various temperatures or, with the addition of suitable heaters/coolers, achieve desired temperature profiles. All these, and other features described herein, permit the present apparatus to be employed in a flexible manner to probe numerous cryopreservation protocols, which is among the chief benefits of this apparatus.

The location of the sample within the pressure chamber and the immersion depth of the chamber into the cooling fluid both are variables affecting the uniformity of cooling. For some of the particular cases described herein we want ice to form on one end and not the other, thus calling for a lack of cooling uniformity. We identify several variables that can affect that uniformity, and others can readily be understood by those with ordinary skills in the art. Thus, some of the protocols we use and describe are largely intended to vary the rate of cooling and biasing. Of course, there is a limit to the amount of biasing that can be introduced. For example, it does not happen in the equipment or tests described herein that 50% of the water can be caused to freeze on one end of the chamber (exerting 210 MPa of pressure) while the other end is at room temperature.

Preliminary Tests

A series of tests were carried out with a view towards evaluating the potential feasibility of the techniques described herein for cryopreservation. Such tests are considered preliminary evaluations only and not to be taken as definitive proof for or against any of the various techniques described herein or techniques used in the tests. However, such preliminary data indicate that, for the test conditions employed, it seems plausible that techniques described herein, or substantial equivalents, could have the potential to provide high yields of viable cryopreserved biosamples in the temperature range around approximately −80 deg. C. without the use of cryoprotectants.

All information provided herein in connection with “Preliminary Tests” is by way of example only and not as a limitation on any equipment, procedures or other technology described herein.

Tests I: Paramecia

Live paramecia in water were used as our biosample. It was noted that the formation of ice caused the paramecia to cease activity, apparently killed by disruption of their structure caused by the formation of ice. In many cases, severe physical disruption was observed, including separation into several distinct fragments. Lack of motion was taken as the indication that the paramecia did not survive. Paramecia movement through the water was taken as an indication that the paramecia survived. In one case, surviving samples were isolated, cultured in media and were observed to reproduce at rates apparently similar to that of control (unprocessed) samples. Of course, this does not indicate the full range of living activities of which the paramecia are capable following processing, but is some evidence that the present approaches merit thorough study and careful consideration.

In summary, the present tests seem to indicate that most paramecia survive in metastable liquid at about −80 deg. C., and under pressure of about 220 MPa, for extended time periods, typically several minutes which was the duration of the typical tests. This conspicuously contrasts with the application of high pressure alone or normal freezing under atmospheric pressure, in which substantially no paramecia survive (precisely none survived in our control tests).

Pressure Chamber

A pressure chamber was constructed from components commercially available from the High Pressure Equipment Company (“HiP”) of Erie, Pa.

-   -   (PC-1) One HiP 40-HM9-4 (in 316 SS) 4″ Coned and Threaded Nipple         Tube, ( 9/16) Outer Diameter, rated for 40,000 psi.     -   (PC-2) Two HiP 40-21HF9-C End Caps (in 316 SS), rated for 40,000         psi.     -   (PC-3) Teflon tape.

The pressure chamber has one end sealed by an end cap that is kept in place throughout the experimental procedures. The other end of the pressure chamber is sealed by an end cap that is opened and closed to load and unload the working fluid (water, in these tests), and the sample chamber. Both end caps were the HiP 40-21HF9-C model noted above. This model end cap comprises three parts, a cap, a tubing collar and a tubing gland. To attach an end cap (PC-2) to a nipple tube (PC-1), first the tubing gland is caused to slide onto the nipple tube, then the tubing collar is screwed onto the nipple tube, and finally the end cap is threaded onto the gland and tightened using a torque wrench to 40-45 ft. lbs. of torque. Teflon tape was used for all threading to help insure a tight seal, reduce possible corrosion at the stainless steel thread contacts, and facilitate unscrewing the parts, among other advantages.

Sample Chamber (or Sample Holder)

Sample holders for these preliminary tests were constructed from parts obtained from the Terumo Medical Corporation of Somerset, N.J.

(SH-1) U-100 (½) cc, 27G×(½)” Insulin syringes.

The syringe parts were modified by the tester as described below.

(SH-2) One shortened tube barrel with both needle and thumb rest removed, crating a sample chamber with a maximum capacity of about 0.5 cc (0.5 ml). One end of this sample chamber SH-2 is capable of receiving a plunger from an insulin syringe SH-1, “Plunger C.” As described below, Plunger C is used to draw the biosample of interest into the sample chamber.

(SH-3) One plunger (“Plunger A”) with a polytetrafluoroethylene (“PTFE”) gasket, trimmed to approximately 1.7″, used to seal the “lower” end of the sample chamber (that is, the end of the sample chamber first inserted into the pressure chamber).

(SH-4) One plunger (Plunger B″) with a PTFE gasket trimmed to approximately 0.7″ used to seal the “upper” end of the sample chamber, and also serving as a spacer between the sample chamber and the upper end cap.

A properly constructed sample holder provides an air-tight and water-tight chamber, permitting Plunger A and Plunger B to move with relatively low frictional resistance (typically less than about 1 Mega Pascal, 1 MPa) to transfer pressure developed within the interior of the pressure chamber, external to the sample chamber, to the interior of the sample chamber containing the biosample of interest, typically in a liquid medium.

Procedures

Loading Sample Chamber

The following procedures are by way of example only, not limitation, as other procedures will be obvious to those with ordinary skills in the art if and when different equipment or components are employed.

(Ld-1) One end of sample chamber SH-2 is inserted into a reservoir containing a solution including the biosample of interest. The sample is drawn into the sample chamber by means of Plunger C inserted into the opposite end of the sample chamber.

(Ld-2) The sample is drawn into the sample chamber as Plunger C is removed from the sample chamber so that the sample chamber tube is filled while still partially submerged in the sample reservoir.

(Ld-3) Plunger A is inserted into the lower (submerged) end of the sample chamber taking care to insure that the gasket is surrounded by liquid when inserted into the sample chamber to avoid the formation of air pockets.

(Ld-4) Plunger A is pushed into the sample chamber, pushing sample out of the upper end of the sample chamber, until the desired quantity of sample remains in the sample chamber.

(Ld-5) Plunger B is inserted into the opposite (upper) end of the sample chamber, insuring that the gasket is surrounded by liquid when inserted to avoid the formation of air pockets.

(Ld-6) Force is applied to Plunger B, causing thereby Plunger A to recede from the center of the sample chamber, until the quantity of biosample within the sample chamber is positioned approximately in the center of the sample chamber, and that both Plunger A and Plunger B extend beyond the sample chamber to serve as spacers from the end cap (Plunger B), and from ice surfaces (Plunger A). Absence of these spacers increases the possibility that proximity of the end cap and/or ice surfaces to the ends of the sample chamber will interfere with the effective transfer of isostatic pressure from the pressure chamber to the interior of the sample chamber.

As a general note, it was found that the spacers can be the site of unanticipated and harmful air pockets so special care should be used to insure such air pockets are not present. Water can be injected into the space between the plunger arm and the sample tube if necessary.

Initial Tests:

The object of initial tests is to investigate in a qualitative manner if cryopreservation with high pressure generated by fluid solidification seems to be feasible. That is, if simple preliminary tests produce no viable cryopreserved biosamples, the foundational concepts of the present approach might be called into question. If, on the other hand, results indicative of moderate viable cryopreservation of samples seem to be present, further investigations would be warranted. Consistent with this initial test philosophy of “keep it simple” the following tests were conducted:

Test Step (TS)-1: Five samples of live paramecia in about 0.06 cc water were prepared. Each sample contained a few (2-5) paramecia, apparently alive and swimming about as observed under magnification.

TS-2: Each sample was placed in a sample chamber entirely filled with water, care being taken to avoid air bubbles within the sample chamber. The actual tests were performed sequentially.

TS-3: Each sample chamber thus prepared is placed in a pressure chamber containing water as the working fluid. Care is taken to insure that the pressure chamber containing the sample chamber is completely filled with water, avoiding air bubbles. The sample chamber is buoyant so it is advantageous to top off the pressure chamber with water while causing the sample chamber to be completely submerged. The pressure chamber is sealed and placed into a liquid nitrogen bath. The experimental variables in these initial tests were the depth to which the pressure chamber is inserted into the liquid nitrogen bath, and the duration the pressure chamber is allowed to reside in the liquid nitrogen, useful variables for facilitating ice formation at the submerged end but avoiding ice nucleation at the center of the tube. Detailed instrumentation to measure pressures and temperatures during the process was not available for these initial tests. Such instrumentation would allow a more careful assessment of the experimental conditions under which viable cryopreservation of biosamples can be achieved. However, tests with various rupture discs indicate that pressures in the range from about 172 MPa to about 250 MPa are achieved.

TS-4: Additionally, two controls were tested, in which a few paramecia were placed in a sample holder and placed in liquid nitrogen, without the use of a pressure chamber. These tests were performed sequentially.

While the thawing process generally represents the inverse of the freezing process, in some ways additional challenges are introduced. Ice typically thaws at a higher temperature than it nucleates, potentially leaving the sample under pressure in a fully thawed state. However, since ice melts at a lower temperature when under pressure, the nucleation temperature is constantly changing as the some portion of the ice thaws and pressure is thereby reduced. Thus, during the warming process, the sample continuously encounters the risk of being below the freeze temperature for a given (reduced) applied pressure. This is in contrast to the cooling process in which the sample is at risk of ice formation only once. In view of the above complication in the warming process, it is typically found to be advantageous to warm the sample side of the pressure container first such that it is always warmer than the ice side so that ice is more likely to form/re-form on the side farthest from the sample.

Results:

Two of the five tests were “unsuccessful” in that no live paramecia were observed following the test procedures. The control tests were similarly “unsuccessful” with no live paramecia observed. However, three of the five tests were “successful” in that 75% to 100% of the paramecia were observed to be alive (swimming) at the conclusion of the pressure, freezing and reanimation processes. These results provide some evidence that processes of the type investigated herein may hold promise for viable cryopreservation of biosamples.

Important points to note from the above include the following:

Pressure created by water on freezing is used to create the pressure applied to one or more sample holders, greatly simplifying and reducing expenses over other pressure-processing techniques.

The apparatus described herein is capable of processing multiple samples in parallel.

It has been observed that the glassy state cooled much below the glass transition temperature tends to shatter. While this may be useful for some approaches to cryofixation, it clearly is not satisfactory for cryopreservation and reanimation. It has been proposed that rapid cooling in typical approaches to glass formation create severe thermal gradients within the sample that leads to cracking. The phased approach presented here creates the glassy phase from metastable supercooled liquid at about −80 deg. C. requiring much less heat transfer to achieve the glass phase and, hence, introduce much less severe thermal gradients. In addition, since the application of elevated pressure suppresses ice formation, there is no need for rapid cooling. As a result, the temperature of the sample can be slowly reduced to reduce or avoid thermal discontinuities.

Tests II: Yeast

Baker's Yeast was selected as a test example to make use of its relatively easy starting and rapid sugar conversion. Its intolerance to high pressure makes this a good test of the benefits of low temperature in combination with high pressure.

A yeast sample was prepared by mixing Baker's Yeast and water in the proportion of about 0.25 teaspoon of Baker's Yeast (4.93 ml) into about 250 ml of water. Three tests were run.

Test II(A)

Five sample holders were prepared, each containing about 2.5 ml of the above yeast/water mixture (yeast sample). Of the five sample holders thus prepared, (IIA-i) was a control (no freezing). (IIA-ii) was used for a flash-freeze test while the remaining holders (IIA-iii)-(IIA-v) were sequentially supercooled generally following the procedures described herein.

Test II(B)

Six sample holders were prepared using the yeast sample as in Test II(A), each sample holder containing about 2.5 ml of the above yeast/water mixture (yeast sample). Of the six sample holders thus prepared, (IIB-i) was a control (no freezing). (IIB-ii) was used for a flash-freeze test while the remaining four holders (IIB-iii)-(IIB-vi) were sequentially supercooled generally following the procedures described herein.

Test II(C)

Six sample holders were prepared using the yeast sample as in Test II(B). Of the six sample holders thus prepared, (IIC-i) was a control (no freezing), while the remaining five holders (IIC-ii)-(IIC-vi) were sequentially supercooled generally following the procedures described herein.

Control Samples:

The control samples remained undisturbed in the sample holder throughout the test procedures performed on the other non-control samples until transferred to yield containers.

Flash Freeze:

The samples in the sample holders for the flash freeze tests were plunged quickly into liquid nitrogen followed by warming by plunging into a warm bath at approximately 98 deg. F.

Supercooling:

The supercooled samples in their sample holders were placed into the pressure chamber substantially as previously described in detail in the previous tests involving paramecia. All samples were cooled to approximately −56 deg. C. and then warmed in the warm bath.

Results:

The yeast from all samples in the test, control, flash freeze and supercooled were placed in containers with about 20 ml wort produced using Coopers Light Dry Malt Extract, pitched, boiled and cooled to produce a wort with about 12% sugar.

The results of the tests (“yield” of viable yeast) was estimated by yeast activity, that is, by the consumption of sugar from the wort. “Yields” are calculated as a percentage of sugar consumed by the yeast relative to that consumed by the control, where the control is assumed to be 100%. The sugar content of the wort was determined by Brix % as measured by a refractometer, deemed to be the most efficient and effective method for sugar determination when, as here, relatively small samples sizes are present.

Since Baker's Yeast was used, the yeast was not able to convert all sugars before succumbing to the alcohol. The process was assumed to be complete when the sugar content had been reduced to approximately 6.9%.

Readings were initially taken hourly until it was determined that the rate of consumption was sufficiently slow such that readings every 4 hours, 24 hours per day were perfectly adequate.

It is found from the above tests that, on average, about 68.5% of the yeast survives flash freezing while about 93% survive using the pressure-freezing combinations described above. That is, the pressure freezing protocols described herein allow almost the same survival of yeast (within about 10%) as if no pressure or cooling has been applied.

Although various embodiments which incorporate the teachings of the present invention have been shown and described in detail herein, those skilled in the art can readily devise many other varied embodiments that still incorporate these teachings. 

1. A method of cryogenically storing a sample so as to reduce sample degradation comprising the concurrent isostatic application of elevated pressure to said sample and cooling said sample, further comprising: a) immersing said sample in a pressure-transmitting medium; b) confining said pressure-transmitting medium in a sample container wherein said sample container transmits external applied pressure to said pressure-transmitting medium and thus to said sample; c) immersing said sample container in a working fluid wherein said working fluid expands upon cooling; d) confining said working fluid with said sample container immersed therein in a pressure-confining container such that expansion of said working fluid will result in elevated pressure being applied to said sample container; and e) cooling said pressure-confining container thereby causing elevated pressure to be applied to said working fluid and said sample container and sample immersed therein.
 2. A method as in claim 1 wherein said pressure-transmitting medium is water.
 3. A method as in claim 1 wherein said working fluid is water.
 4. A method as in claim 1 wherein said pressure-transmitting container is a substantially rigid tube having sliding gaskets on one or more ends thereof capable of transmitting elevated pressure from said working fluid to said pressure-transmitting medium.
 5. A method as in claim 1 wherein said cooling of said pressure-confining container is performed preferentially on selected portions of said pressure-confining container.
 6. A method as in claim 1 wherein said sample container can be moved to different positions within said pressure-confining container.
 7. A method as in claim 1 further comprising two or more of said sample containers immersed in said working fluid confined in said pressure-confining container.
 8. A method of producing metastable supercooled liquid in a sample without the necessity of an ultra-rapid cooling step, comprising: a) immersing said sample in a pressure-transmitting medium; b) confining said pressure-transmitting medium in a sample container wherein said sample container transmits external applied pressure to said pressure-transmitting medium and thus to said sample; c) immersing said sample container in a working fluid wherein said working fluid expands upon cooling; d) confining said working fluid with said sample container immersed therein in a pressure-confining container such that expansion of said working fluid will result in elevated pressure being applied to said sample container; and e) cooling said pressure-confining container thereby causing elevated pressure to be applied to said working fluid and said sample container and sample immersed therein until a metastable liquid phase is produced in said sample.
 9. A method as in claim 8 wherein said pressure-transmitting medium is water, said working fluid is water, said cooling is to a temperature of about −80 deg. C. thereby producing pressures on said sample approximately in the range from about 200 MPa to about 220 MPa.
 10. A method of producing a state of matter substantially similar to high density amorphous water in a water-containing sample without the necessity of an ultra-rapid cooling step, comprising: a) immersing said sample in a pressure-transmitting medium; b) confining said pressure-transmitting medium in a sample container wherein said sample container transmits external applied pressure to said pressure-transmitting medium and thus to said sample; c) immersing said sample container in a working fluid wherein said working fluid expands upon cooling; d) confining said working fluid with said sample container immersed therein in a pressure-confining container such that expansion of said working fluid will result in elevated pressure being applied to said sample container; e) cooling said pressure-confining container thereby causing elevated pressure to be applied to said working fluid and said sample container and sample immersed therein until a metastable liquid phase is produced in said sample; and f) continuing cooling until a temperature at or below the glass transition temperature of said sample is achieved.
 11. A method as in claim 1 further comprising: f) preferentially warming the sample side of said pressure-confining container thereby recovering said sample from cryogenic storage. 